Supplementary information
Protoilludane sesquiterpenoids as scaffold structures for new
antimicrobials against Mannheimia haemolytica
Gemma Assante1, Sabrina Dallavalle2, Piera Anna Martino3
Growth condition of Echinodontium tsugicola
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Antimicrobial tests
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General chemical experimental methods
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Preparation of compounds 16-19
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DISAA, Dipartimento di Scienze Agrarie ed Agroambientali – Produzione, Territorio e
Agroenergia, Università di Milano, Italy
2
DeFENS, Dipartimento di Scienze per gli Alimenti, la Nutrizione e l’Ambiente, Università di
Milano, Italy
3
Dipartimento di Scienze Veterinarie e Sanità Pubblica, Università di Milano, Italy
E-mail: sabrina.dallavalle@unimi.it
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Growth condition of Echinodontium tsugicola
Echinodontium tsugicola CBS 248.51 and Echinodontium tsugicola kindly supplied by
Dr. Yoshihito Shiono (Department of Bioresource Engineering, Faculty of Agriculture,
Yamagata University, Tsuruoka, Yamagata, Japan)., were cultivated on a broth obtained
filtering boiled potatoes and carrots (PC broth, 100 g each L-1) and enriched with 2%
glucose. Still cultures were carried out for three weeks at 22 °C in the dark in Roux flasks
(750 cm3) with 100 ml broth and this volume/surface ratio gave the best results. The
preculture consisted of Erlenmeyer flasks (250 ml) containing 80 ml nutrient broth (malt
extract, peptone, glucose 2:0,2:2%, respectively) inoculated with the mycelium scarified
from a Petri dish (9 cm diam) of the same medium with agar where the fungus was grown
for two weeks. Pre-culture Erlenmeyer flasks were incubated on an orbital shaker at 150
rpm for 72 h and then used to inoculate the Roux flasks (10 ml).
Antimicrobial tests
MIC determination was performed using broth dilution in 96-wells plate. Strains of M.
haemolytica (Strain ATCC 14003) were isolated on blood-agar plates (Oxoid, Italy) and
incubated at 37 °C for 48 hours in microphilic atmosphere (5% CO2). The isolates were
maintained in BHI slants (Brain-Heart Infusion Agar, Oxoid, Italy) until their use in the
microdilution test. Compounds were weighted and dissolved in DMSO to obtain a final
concentration of 256 g/mL (initial solution; 2x concentration). 100 L of Mueller
Hinton Broth (Oxoid, Italy) were dispensed into all wells; then 100 L of the 2x
antibiotic solutions were added into the wells in column 1 (far left of plate). After mixing
this suspension in colum1, 100 L were withdrawn from column 1 to column 2; this
makes column 2 a twofold dilution of column 1. The procedure was repeated down to
column 10; column 11 was positive to bacterial control and column 12 negative. The
appropriate bacterial inoculum was 105 CFU/mL and 5 L were added to each well from
column 11 to 1. Then the plate was incubated at 37 °C for 48 hours in microphilic
atmosphere.
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After the incubation time we have read the plate; MIC (expressed as g/mL), is the
lowest concentration of a drug that can inhibit the bacterial growth (MIC50)
All the tests were performed twice for all the tested molecules.
The same procedures was followed for MIC determination of Escherichia coli (ATCC
25922) and Pasteurella multocida (ATCC 15743) but for E. coli we incubated the plate
in aerobic conditions for 24 hours at 37 °C.
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General chemical experimental methods
All reagents and solvents were reagent grade or were purified by standard methods before
use. Melting points were determined in open capillaries and are uncorrected. NMR
spectra were recorded at 300 MHz. Chemical shifts (
values) are given in ppm and Hz, respectively. Solvents were routinely distilled prior to
use; anhydrous tetrahydrofuran (THF) and ether (Et2O) were obtained by distillation from
sodium-benzophenone ketyl; dry methylene chloride was obtained by distillation from
phosphorus pentoxide. All reactions requiring anhydrous conditions were performed
under a positive nitrogen flow, and all glassware were oven dried and/or flame dried.
Isolation and purification of the compounds were performed by flash column
chromatography on silica gel 60 (230-400 mesh). Analytical thin-layer chromatography
(TLC) was conducted on Fluka TLC plates (silica gel 60 F254, aluminium foil).
Compound 16 9
To a solution of Tsugicoline A (25 mg, 0.094 mmol) in methanol (1.5 mL) TEA (0.05
mL, 0.36 mmol) was added. The solution turned from yellow to dark orange. After
stirring for 4 h at room temperature, the solvent was evaporated under reduced pressure
and the crude was purified by plc (CH2Cl2/MeOH 15:1) to obtain 10 mg (43%) of
compound 15 as an oil.
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H NMR (acetone-d6) 9.48 (1H, s); 6.78 (1H, s); 5.14 (1H, d, J = 7.8 Hz); 4.71 (1H, d, J =
7.8 hz); 3.16 (1H, m); 2.65 (1H, ddd, J = 12.1; 7.0; 7.5 Hz); 2.10 (1H, m); 1.70 (1H, dd, J
= 13.0, 2.0 Hz); 1.55 (1H, m); 0.95-1.20 (10H, m).
Compound 17 17
To a solution of Tsugicoline A (100 mg, 0.38 mmol) in methanol (5 mL) NaBH4 (20 mg,
0.53 mmol) was added portionwise at room temperature. The solution was stirred for 2h
then water (5ml) and a few drops of 2M HCl were added. Methanol was evaporated and
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the aqueous phase was extracted with ethyl acetate. The organic layer was dried filtered
and the solvent was evaporated. The crude was purified by PLC (CH2Cl2/ MeOH 15:1) to
obtain 16 (80 mg, 79%) and a small amount (3 mg) of its epimer. Mp 85-87°C 1H NMR
(acetone-d6)  4.60 (1H, s); 4.45 (2H, s); 3.90-4.10 (2H, m); 3.65 (1H, m); 2.10-2.30 (2H,
m); 1.78 (1H, dd, J = 12.6, 7.2 Hz); 1.30-1.40 ( 2H, m); 1.10-1.20 (1H, m); 1.08 (3H, s);
1.00 (3H, s); 0.97 (3H, s).
Compound 18 9
Tsugicoline A (100 mg, 0.38 mmol) was dissolved in dry pyridine (1 mL) and added with
acetic anhydride (2 ml). The solution was stirred at 0°C for 6h. The mixture was then
poured into iced water and the aqueous phase was extracted with ethyl acetate (3 x 5 ml)
The collected organic phases were washed twice with 1N HCl and with a saturated
solution of NaCl, dried and evaporated to obtain 110 mg of a brown oil. Purification by
PLC (hexane: ethyl aceate 2:1) afforded 60 mg (41%) of the title compound as a
colorless oil. 1H NMR (acetone-d6)  5.40 (1H, d, J = 9.2 Hz); 5.10 (1H, s); 4.84 (1H, d, J
= 16 Hz); 4.69 (1H, d, J = 16 Hz); 2.63-2.67 (1H, m); 2.46-2.50 (1H, m); 2.12 (6H, s);
2.10 (3H, s); 1.65-1.67 (2H, m); 1.42 (1H, dd, J = 10.6, 13 Hz); 1.17-1.21 (1H, m); 1.07
(3H, s); 0.99 (6H, s).
Compound 19a 18
To a solution of Tsugicoline A (50 mg, 0.19 mmol) in MeOH (13 ml), NH2OH.HCl (25
mg, 0.38 mmol) and NaH2PO4 (25 mg, 0.21 mmol) were added. The solution was stirred
at room temperature overnight, then the solvent was evaporated. The crude was purified
by PLC (CH2Cl2/MeOH 9:1) to obtain compound 18a as a mixture 1:1 of the two oximes
(37 mg, 74%). Mp> 270°C; 1H NMR (acetone-d6) (values in parentheses refer to the
second isomer) 4.67 (4.43) (1H, d, J = 13.0 Hz); 4.54 (4.32) (1H, d, J = 13.0 Hz); 4.36
(4.51) (1H, s); 4.25-4.29 (1H, m); 2.36-2.40 (1H, m); 2.30-2.36 (1H, m); 1.75-1.85 (1H,
m); 1.35-1.50 (2H, m); 1.20-1.26 (1H, m); 1.10 (3H, s); 0.99 (6H, s).
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Compound 19b
To a solution of Tsugicoline A (20 mg, 0.08 mmol) in MeOH (5 ml), NH2OCH3.HCl (13
mg, 0.16 mmol) and NaH2PO4 (10 mg, 0.1 mmol) were added. The solution was stirred at
room temperature for 2h, then the solvent was evaporated under vacuum without heating.
The crude was added with waterand the aqueous phase was extracted with ethyl acetate
(3 x 10 ml). The organic layer was dried and evaporated to give the title compound 18b
as a mixture 1:1 of the two oximes (15 mg, 68%). Mp> 270°C; 1H NMR (acetone-d6)
(values in parentheses refer to the second isomer) 4.80 (4.70)(1H, brs); 4.56 (4.43) (1H,
d, J = 13.0 Hz); 4.20-4.50 (3H, m); 3.85 (3.80) (3H, s); 2.22-2.42 (2H, m); 1.75-1.85 (1H,
m); 1.35-1.50 (2H, m); 1.20-1.30 (1H, m); 1.10 (3H, s); 0.99 (6H, s).
Compound 19c
To a solution of Tsugicoline A (50 mg, 0.19 mmol) in MeOH (13 ml), NH2OCH2Ph.HCl
(59 mg, 0.37 mmol) and NaH2PO4 (25 mg, 0.21 mmol) were added. The solution was
stirred at room temperature for 3h, then the solvent was evaporated under vacuum
without heating. The crude was added with waterand the aqueous phase was extracted
with ethyl acetate (3 x 10 ml). The organic layer was dried and evaporated to give the
title compound 18b as a mixture 1:1 of the two oximes 62 mg, 89%). Mp> 270°C; 1H
NMR (acetone-d6) (values in parentheses refer to the second isomer)  7.20-7.45 (5H, m);
5.15 (5.05) (2H, s); 4.80 (4.75)(1H, brs); 4.62 (4.53) (1H, d, J = 13.0 Hz); 4.20-4.55 (3H,
m); 2.20-2.45 (2H, m); 1.75-1.85 (1H, m); 1.35-1.50 (2H, m); 1.15-1.30 (1H, m); 1.10
(3H, s); 0.95 (6H, s).
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